ADAMTSL2 is a potential prognostic biomarker and immunotherapeutic target for colorectal cancer: Bioinformatic analysis and experimental verification

The ADAMTS Like 2 (ADAMTSL2) mutation has been identified to be associated with different human genetic diseases. The role of ADAMTSL2 is unclear in colorectal cancer (CRC). The study investigated the expression of ADAMTSL2 in both pan cancer and CRC, using data from The Cancer Genome Atlas (TCGA) database to assess its diagnostic value. The study examined the correlation between ADAMTSL2 expression levels and clinical characteristics, as well as prognosis in CRC. The study explored potential regulatory networks involving ADAMTSL2, including its association with immune infiltration, immune checkpoint genes, tumor mutational burden (TMB) / microsatellite instability (MSI), tumor stemness index (mRNAsi), and drug sensitivity in CRC. ADAMTSL2 expression was validated using GSE71187 and quantitative real-time PCR (qRT-PCR). ADAMTSL2 was aberrantly expressed in pan cancer and CRC. An increased level of ADAMTSL2 expression in patients with CRC was significantly associated with the pathologic N stage (p < 0.001), pathologic stage (p < 0.001), age (p < 0.001), histological type (p < 0.001), and neoplasm type (p = 0.001). The high expression of ADAMTSL2 in patients with CRC was found to be significantly associated with a poorer overall survival (OS) (HR: 1.67; 95% CI: 1.18–2.38; p = 0.004), progression-free survival (PFS) (HR: 1.55; 95% CI: 1.14–2.11; p = 0.005) and disease-specific survival (DSS) (HR: 1.83; 95% CI: 1.16–2.89; p = 0.010). The expression of ADAMTSL2 in patients with CRC (p = 0.009) was identified as an independent prognostic determinant. ADAMTSL2 was associated with extracellular matrix receptor (ECM-receptor) interaction, transforming growth factor β (TGF-β) signaling pathway, and more. ADAMTSL2 expression was correlated with immune infiltration, immune checkpoint genes, TMB / MSI and mRNAsi in CRC. ADAMTSL2 expression was significantly and negatively correlated with 1-BET-762, Trametinib, and WZ3105 in CRC. ADAMTSL2 was significantly upregulated in CRC cell lines. The high expression of ADAMTSL2 is significantly correlated with lower OS and immune infiltration of CRC. ADAMTSL2 may be a potential prognostic biomarker and immunotherapeutic target for CRC patients.


Introduction
Colorectal cancer (CRC), which affects the colon and rectum, is the third most prevalent cause of cancer-related mortality globally, accounting for more than 1.85 million incidences and 850,000 fatalities annually [1].These findings align with data from the National Cancer Center, which reports a 5-year survival rate of approximately 50% for CRC in China [2].CRC is a heterogeneous disorder with different pathogenic mechanisms that involve somatic mutations, gene fusion, genetic instability, and epigenetic alterations [3].Despite the tremendous efforts made in the treatment of CRC in terms of early diagnosis and multidisciplinary treatment, including improved screening methods, surgical procedures, chemotherapy, radiation therapy, targeted biologic therapy, and immunotherapy, a significant proportion of patients with CRC, especially those with advanced CRC, still have a poor prognosis [4].The 5-year survival rate for patients with early-stage CRC is approximately 90%, and once a patient is diagnosed with advanced CRC, the survival rate drops to 13.1% [5].With the development of genome sequencing technology, increasing biomarkers that predict tumor development and prognosis are being discovered.Therefore, it is necessary to explore potential prognostic biomarkers that facilitate the early diagnosis and treatment of CRC.
ADAMTS Like 2 (ADAMTSL2) is a protein coding gene.This gene is responsible for encoding a constituent of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) and the ADAMTS-like protein family [6,7].Mutations in ADAMTSL2 have been found in different human genetic disorders [8][9][10].Univariate analysis showed that ADAMTSL2 expression was correlated with the prognosis of hepatocellular carcinoma (HCC) [11].However, the clinical implications and regulatory network of ADAMTSL2 in patients with CRC remain uncertain.
The objective of this study was to investigate the different expression patterns of ADAMTSL2 in pan cancer and CRC, and to assess the diagnostic importance of ADAMTSL2 in CRC using data from The Cancer Genome Atlas (TCGA) database.Furthermore, we examine the association between ADAMTSL2 expression levels and clinical characteristics, as well as prognosis in CRC.To elucidate the potential regulatory network of ADAMTSL2, genomic enrichment analysis (GSEA) was employed.Furthermore, we investigate the correlation between ADAMTSL2 and immune infiltration, immune checkpoint genes, tumor mutation burden (TMB), microsatellite instability (MSI), tumor stemness index (mRNAsi), and drug sensitivity in CRC.Additionally, we utilized the Gene Expression Omnibus (GEO) database (GSE71187) to validate the abnormal expression of ADAMTSL2.This study has identified a potentially valuable prognostic biomarker and immunotherapeutic target for patients with CRC.

Materials and methods
The data utilized in this study were sourced from the TCGA and GEO databases.Given that these databases provide free access to data for research and publication purposes, our study did not require the ethical approval or consent of the patient.It is important to note that this study used only cell lines and did not involve the use of human or animal tissues.

Sample collection
Pan-cancer patients were collected from TCGA database [12].A total of 647 CRC tissues and 51 adjacent normal tissues were obtained from patients with CRC (colon & rectal cancer, COAD&READ) [13,14].Furthermore, 157 CRC tissues and 32 normal tissues were collected from the GSE71187 dataset.

Differential expression of ADAMTSL2
The R software, ggplot2, and statistical methods (stats [4.2.1] and car) were used for the analysis of the unpaired, paired, and pan-cancer sample.
ROC analysis was performed using the R software and the pROC package, as well as the ggplot2 package [16].Clinical variables compared tumor and normal samples.

Correlation of ADAMTSL2 with clinical characteristics
We used the R software and ggplot2 to explore the correlation between ADAMTSL2 expression and clinical characteristics.Clinical variables included N stage, pathologic stage, age, histological type, and neoplasm type.
The R software and the dichotomous logistic model were used for logistics analysis [17].The dependent variable was ADAMTSL2.The types of independent variables were low and high dichotomous.

Correlation between ADAMTSL2 and prognosis
Kaplan-Meier analysis was performed using R software, specifically using the survminer package [3.3.1] and the survival package [3.3.6] [18].We divided the expression levels of ADAMTSL2 into high and low groups.The subgroups were classified into two categories, namely 0-50 and 50-100.The types of prognosis considered were overall survival (OS) and disease-specific survival (DSS).Further prognostic data were obtained from reference [19].
Cox regression analysis was performed using the R software, using the survivor package and the Cox regression module.The prognosis of interest was OS.The clinical characteristics of the variables and ADAMTSL2 were included in the analysis, together with additional prognostic data obtained from a reference source [19].
The R software and the ggplot2 package were used for the forest plot.The rms package & survival package and Cox were used for the nomogram plot.The prognosis considered in this study was OS.Variables taken into account were T stage, N stage, pathological stage, age, and ADAMTSL2.We collect prognostic data from the reference [19].

ADAMTSL2-associated pathways
The R software and deseq2 were used for differential analysis of a single gene [20].The high and low expression groups were defined as 0-50% and 50-100%, respectively.
We used R software in conjunction with the ggplot2 and clusterProfiler packages to perform GSEA [21,22].The species selected for the analysis was Homo sapiens, and the reference gene collection used was c2.cp.v7.2.symbols.gmt[Curated].The gene set database used was MSigDB Collections, which can be accessed through the provided hyperlink and includes comprehensive descriptions of each individual gene set.The significance conditions were generally as follows: P.adj < 0.05 & FDR < 0.25.

Correlation of ADAMTSL2 with immune infiltration and immune checkpoint-related genes
The immunocell algorithm was applied using ssGSEA, which is an algorithm integrated within the GSVA package [23,24].We obtained 24 markers of immune cells from the reference [25].The statistical method is Spearman.
After grouping the main variables, we select appropriate statistical methods (stats package and car package) based on the characteristics of the data format for statistical analysis (if the statistical requirements are not met, statistical analysis will not be conducted) and use the ggplot2 package to visualize the data.We calculate the stromal and immune scores of the corresponding cloud data using R package estimate [1.0.13] [26].The statistical method is the Wilcoxon rank sum test.
The RNAseq data (level 3) and the corresponding clinical information of CRC were obtained from TCGA.In this study, the expression of eight immune checkpoint genes, namely, sialic acid binding Ig like lectin 15 (SIGLEC15), Indoleamine 2,3-dioxygenase 1 (IDO1), CD274, Hepatitis A virus cellular receptor 2 (HAVCR2), programmed cell death 1 (PDCD1), cytotoxic T-lymphocyte associated protein 4 (CTLA4), lymphocyte activating 3 (LAG3), and programmed cell death 1 ligand 2 (PDCD1LG2), was analyzed.We analyze the expression of these eight genes.We extracted the expression values of these 8 genes and observed the expression of genes related to immune checkpoint.We used statistical analysis using R software v4.0.3.If not specified otherwise, the rank sum test detects the difference between the two groups of data, and a P-value<0.05 is considered statistically significant.

The relationship between ADAMTSL2 and TMB/MSI
TMB, an abbreviation for tumor mutational burden, serves as a quantifiable metric for the number of mutations observed in cancer [27,28].MSI is a phenotype that occurs due to malfunction of DNA repair mechanisms and is present in approximately 15% of CRC [29].RNAseq data (level 3) and the corresponding clinical information for CRC were obtained from TCGA.We used Spearman's correlation analysis to describe the correlation between quantitative variables without a normal distribution.A p-value less than 0.05 is considered statistically significant.

The relationship between ADAMTSL2 and mRNAsi
The OCLR algorithm is used to calculate mRNAsi [30].Based on mRNA expression characteristics that include a gene expression profile containing 11774 genes, we used the same spearman correlation (RNA expression data) and then mapped the stemness index to the range of [0,1] using a linear transformation by subtracting the minimum and dividing by the maximum [31].All the above analysis methods and the R software packages were executed using the version v4.0.3 of the R software (R Foundation for Statistical Computing, 2020).

Expression of ADAMTSL2 in CRC single cells
The Tumor Immune Single Cell Hub 2 (TISCH2) (http://tisch.comp-genomics.org/)database is a scRNA-seq database that focuses primarily on the tumor microenvironment (TME).TISCH2 offers a comprehensive cell-type annotation at the single-cell level, facilitating the investigation of TME in various types of cancer.

Genomic variants of ADAMTSL2 in CRC patients
The RNAseq data (level3), the mutation maf data and the corresponding clinical information for CRC were obtained from the TCGA dataset (https://portal.gdc.com).Somatic mutations in CRC patients were downloaded and visualized using the maftools package in R software.Horizontal histograms showed a high frequency of mutations in patients with CRC.

Drug sensitivity of ADAMTSL2 in pan cancer
ADAMTSL2 drug sensitivity was analyzed in pan cancer using the RNAactDrug database available at http://bio-bigdata.hrbmu.edu.cn/RNAactDrug/index.jsp.

Validation of ADAMTSL2 gene expression in GEO database
In order to improve the reliability of the TCGA database, data pertaining to CRC samples were obtained from the GEO database.GSE71187 contained 157 CRC tissues and 32 normal tissues.We used GSE71187 for the analysis of the expression of the ADAMTSL2 gene.

Statistics analysis
Statistical analysis was performed using the R software.It is important to note that a p-value of less than 0.05 was considered to indicate statistical significance in this analysis.

ADAMTSL2 is aberrantly expressed in pan-cancer and CRC
As shown in S1

Association of ADAMTSL2 with clinical characteristics in patients with CRC
ADAMTSL2 expression was found to be significantly associated with the pathologic N stage (p < 0.001), pathologic stage (p < 0.001), age (p < 0.001), histological type (p < 0.001), and neoplasm type (p = 0.001) in patients with CRC (S1, S2 Tables and Fig 2).

ADAMTSL2-related pathways
We identified 45 sets of genes significantly differentially enriched in the expression phenotype of ADAMTSL2 by GSEA analysis.The analysis of the datasets revealed that extracellular matrix receptor (ECM-receptor) interaction, transforming growth factor β (TGF-β) signaling pathway, and more, were among the top 9 datasets with low P values (Fig 5).In READ patients, ADAMTSL2 expression was significantly and positively correlated with PDCD1 and SIGLEC15 expression (Fig 7).In patients with COAD, a positive correlation was observed between the expression of ADAMTSL2 and SIGLEC15, while a negative correlation was found with the expression of CD274, HAVCR2, LAG3 and TIGIT (Fig 7).The results suggested that ADAMTSL2 was associated with several immune checkpoint genes in CRC.

Correlation between the expression of ADAMTSL2 and TMB/MSI
In CRC, the expression of ADAMTSL2 showed a significant negative correlation with TMB (p = 6.

Correlation between the expression of ADAMTSL2 and mRNAsi
Cancer progression involves progressive loss of differentiated phenotype and the acquisition of progenitor / stem cell-like features.The expression of ADAMTSL2 in CRC showed a negative correlation with mRNAsi (Fig 9).

ADAMTSL2 expression in single CRC cells was correlated with immune infiltration
As shown in Fig 10 , ADAMTSL2 was up-regulated in multiple individual CRC cells, including CD8Tex, endothelial, fibroblasts, and myofibroblasts.

ADAMTSL2 expression was correlated with drug sensitivity
The present study used the RNAactDrug database to investigate the potential link between ADAMTSL2 expression and drug sensitivity.Our findings indicated a positive correlation  1).On the contrary, the study revealed a negative correlation between ADAMTSL2 expression and the sensitivity of T0901317, Vorinostat, XMD14-99, 9-nitro-10-methoxy-20(s)-camptothecin and thalphenine chloride (Table 1).These results suggested a possible association between ZP3 and drug resistance to some drugs.In this study, the expression of ADAMTSL2 was higher in CRC tissues compared to normal colorectal tissues (p < 0.001).The high expression of ADAMTSL2 was associated with N stage (p < 0.001), pathologic stage (p < 0.001), age (p < 0.001), histological type (p < 0.001), and neoplasm type (p = 0.001).In patients with CRC, ADAMTSL2 expression was higher in patients with N1 & N2 stage compared to patients with N0 stage.In patients with CRC, ADAMTSL2 expression was higher in patients with stages III & IV compared to patients with stages I & II.These suggested that ADAMTSL2 may be involved in tumorigenesis and progression.ADAMTSL2 was correlated with poor OS (p = 0.004), PFS (p = 0.005) and DSS (p = 0.010) in patients with CRC.The expression of ADAMTSL2 (p = 0.009) has been identified as an autonomous prognostic determinant in patients with CRC.

As shown in
ADAMTSL2 was found to improve myoblast differentiation by augmenting WNT signaling [38].Furthermore, up-regulation of the ECM glycoprotein ADAMTSL2 was observed in heart failure, which was found to inhibit TGF-β signaling in cardiac fibroblasts [39].This investigation has revealed an association between ADAMTSL2 and various pathways, including the ECM-receptor interaction, TGF-β signaling pathway, and more.The interactions between ECM and cellular receptors constitute one of the crucial pathways involved in the progression and metastasis of CRC [40].Curcumin may inhibit LG5 (+) CRC by inducing autophagy and inhibiting the carcinogenic TFAP2 mediated ECM pathway [41].TGF-β signaling is important   in the context of inflammation and tumorigenesis by modulating cell growth, differentiation, apoptosis, and homeostasis [42].The specific mechanism by which ADAMTSL2 mediates the occurrence of CRC through these pathways needs further investigation.
Examining immune infiltration in CRC has emerged as a prominent area of research [43].Immunotherapy has achieved strong antitumor efficacy in many cancers [44].Other immunotherapies for CRC patients are still being developed, despite the good efficacy of immune checkpoint inhibitors (ICI) [45].This study aimed to investigate the relationship between ADAMTSL2 expression and immunity in CRC.The expression of ADAMTSL2 in CRC was found to be correlated with the infiltration of various types of immune cells, including aDC, cytotoxic cells, T cells, T helper cells, Th1 cells, Th2 cells, Eosinophils, iDC, Mast cells, NK CD56dim cells, NK cells, pDC, TFH, and TReg.These observed associations may imply potential mechanisms by which ADAMTSL2 hinders the functioning of aDC, cytotoxic cells, T cells, T helper cells, Th1 cells, and Th2 cells, while improving the function of Eosinophils, iDC, Mast cells, NK CD56dim cells, NK cells, pDC, TFH, and TReg.ADAMTSL2 expression in patients with CRC is associated with CD274, HAVCR2, LAG3, PDCD1, SIGLEC15 and TIGIT.There is a significant positive correlation between the expression level of ADAMTSL2 and the stromal score.This indicates that ADAMTSL2 can affect the CRC microenvironment by altering stromal cells.The specific mechanism between ADAMTSL2 and immune infiltration and immune checkpoints in CRC needs to be further investigated.
More than 80% of MSI high tumors have a TMB value of >20 mutations/Mb [27].Furthermore, the study examined the correlation between ADAMTSL2 expression and MSI or TMB.The results revealed a significant association between ADAMTSL2 expression and MSI and TMB in CRC.ADAMTSL2 in CRC is negatively correlated with TMB and MSI, indicating that ADAMTSL2 may reflect the cancer immunogenicity of CRC.The influence of ADAMTSL2 expression on the response of CRC patients to immune checkpoint therapy suggested the potential to use immunotherapy in CRC treatment guided by ADAMTSL2.
Cancer stem cells (CSCs) propose that CSCs are central to carcinogenesis [46].mRNAsi is an index calculated based on expression data [47].In this study, we found that ADAMTSL2 expression is negatively correlated with mRNAsi in CRC.The specific mechanism between ADAMTSL2 and CSC in CRC needs further study.
However, there are some limitations to this study.First, this study is a clinical significance and regulatory network analysis of ADAMTSL2 in CRC using public databases TCGA and GEO and these results need to be further validated in real-world samples.Second, the molecular mechanism of ADAMTSL2-mediated CRC development needs to be further experimentally validated.

Fig 4 .Fig 5 .Fig 6 .
Fig 4. ADAMTSL2 was an independent variable for predicting OS in CRC.(A) Forest plot display of the results of the multifactorial Cox regression analysis of ADAMTSL2 and clinical characteristics in CRC.(B) Nomograms were constructed to predict the probability of OS at 1-, 3-, and 5-years in CRC.https://doi.org/10.1371/journal.pone.0303909.g004

Fig 8 .Fig 9 .
Fig 8. ADAMTSL2 expression in CRC was associated with TMB/MSI.(A) TMB.(B) MSI.Horizontal coordinates represent the gene and TMB/MSI correlation coefficients.Vertical coordinates on the graph correspond to various tumors, while the dot sizes represent the magnitude of the correlation coefficients.Furthermore, the colors employed in the schematic indicate the significance of the p-values, with bluer shades indicating smaller p-values.https://doi.org/10.1371/journal.pone.0303909.g008

Fig 11 .
Fig 11.Somatic mutations of ADAMTSL2 in CRC.(A) Lollipop plot showing the distribution of mutations of the ADAMTSL2 gene.(B) Oncoplot showing the somatic landscape of ADAMTSL2 in the CRC cohort.Genes are sorted by mutation frequency and samples are sorted by disease histology, as indicated in the annotation column (bottom).The sidebar plot shows the transformed log10 Q values estimated by MutSigCV.Mutation information for each gene in each sample is shown in the waterfall plot, where the different colors with specific annotations at the bottom indicate the various types of mutations.The vignettes above the legend show the number of mutation burdens.(C) Cluster summary plots show the distribution of variants according to variant classification, type, and SNV category.The bottom (from left to right) indicates the mutational load for each sample (variant classification type).The stacked bar graph shows the top ten mutated genes.https://doi.org/10.1371/journal.pone.0303909.g011